Evaluation and comparison of hemagglutination inhibition and indirect immunofluorescence tests for the detection of antibodies against duck Tembusu virus.

Currently, duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is widely spread and becomes endemic in duck populations in Asia, causing significant economic losses in the duck producing industry. To early detection and control of DTMUV, the well-validated diagnostic tests for efficient detection of DTMUV infection in ducks are needed. In this study, we validated and compared hemagglutination inhibition (HI) and indirect immunofluorescence (IFA) tests for identifying antibodies against DTMUV in duck serum samples. Our results demonstrated that HI and IFA tests can both be used to detect antibodies against DTMUV in duck serum samples with high sensitivity (100%), specificity (>87%) and overall agreement with the gold standard serum neutralization (SN) test (>90%). Additionally, DTMUV-specific antibody titres determined by HI and IFA tests correlated well with the neutralizing antibody titres obtained by SN test. No cross-reactivity against common duck viruses and other flaviviruses was observed in both tests. It is interesting to note that HI test had higher diagnostic specificity and exhibited a stronger positive correlation with SN test than IFA test. Evaluating the performance of HI and IFA tests with experimental and field serum samples revealed that both tests showed comparable performance with SN test in terms of antibody kinetic and detection rate. Collectively, these findings support the use of both tests, particularly HI test, as the alternative to SN test for measuring the antibody responses against DTMUV in ducks. These tests could be the suitable choices for DTMUV diagnosis, epidemiological study and vaccine efficacy evaluation.

Antinuclear antibodies (ANAs) detected by indirect immunofluorescence (IIF) method in acute COVID-19 infection; future roadmap for laboratory diagnosis.

INTRODUCTION: As in other viral infections, anti-nuclear antibodies (ANAs) are observed in SARS-CoV-2 infection. We investigated the presence of autoantibodies in acute COVID-19 and the association with early laboratory findings. MATERIALS AND METHODS: We examined 50 sera (>18 years, 25 Female) from patients with acute COVID-19. ANAs (HEp-20-10 liver biochip), anti-neutrophil cytoplasmic antibody (ANCA, Europlus Granulocyte Mosaic 32) and anti-double stranded DNA were investigated with product of Euroimmune AG (Luebeck, Germany) by indirect immunofluorescence (IIF) method. Also, antibody against cyclic citrullinated peptide (anti-CCP) was examined by a chemiluminisens assay (Euroimmun AG, Luebeck, Germany). Samples from 50 blood bank donors collected before the COVID-19 pandemic were used as controls. RESULTS: The IIF-ANA test was positive in 18% (N = 9/50) of the patients. The median time of sample collection was 7 days (range: 1-28 days) after diagnosis. ANA was positive in only one (2%) control sample. Five (55.5%) patients were ANA positive with a strong titer (3+). There was no relationship between antibody titration and time of sample collection (p = 0,55). Anti-CCP was detected in a nucleolar (3+) positive patient (2%). ANA was detected in 14.28% (N = 1/7, rods-rings (±), p = 0,78) of patients in the intensive care unit(ICU). Patients treated in the clinic have more and higher titers of ANA, mostly in nucleolar patterns, than ICU patients. CONCLUSIONS: The variety of antibodies detected in acute COVID-19 and the uncertainty of how long they persist can lead to confusion, especially in the diagnosis of systemic autoimmune rheumatic diseases for IIF-ANA testing in immunology laboratories. Improvements in cell lines and methods will facilitate the diagnostic process.

Serological Survey on the Occurrence of Rickettsia spp., Neospora caninum, Bartonella henselae and Toxoplasma gondii in Cats from Tuscany (Central Italy).

Asymptomatic cats often harbor pathogens, some of which have not been largely investigated in feline populations. The aim of this study was to evaluate the occurrence of antibodies against Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Neospora caninum, Bartonella henselae and Toxoplasma gondii in cats from Tuscany. Ninety-five blood serum samples, previously collected, were analyzed by indirect immunofluorescence assay. Fifty-six (58.94%) cats had antibodies to at least one investigated pathogen: 28 (29.47%) cats were positive for B. henselae, 17 (17.89%) for R. felis, 14 (14.73%) for R. conorii, 14 (14.73%) for T. gondii, 2 (2.1%) for N. caninum. No cats were positive for R. typhi. Positive reactions to two or more pathogens were detected in 18 (18.94%) cats. The occurrence of antibodies against these microorganisms suggests that cats, even though asymptomatic, may be infected by pathogens, often zoonotic, and thus may be a source of infections for other animals and humans.

Determinación de anticuerpos anti-dsDNA: evaluación del desempeño de un inmunoensayo quimioluminiscente y correlación con el método de inmunofluorescencia indirecta / Detection of anti-dsDNA antibodies: evaluation of performance of a chemiluminescent immunoassay and correlation to the indirect immunofluorescence assay / Determinação de anticorpos anti-dsDNA: avaliação do desempenho de um imunoensaio quimioluminiscente e correlação com o método de imunoflourescência indireta

Resumen La determinación de anticuerpos anti-dsDNA es de utilidad para el diagnóstico y seguimiento clínico de pacientes con lupus eritematoso sistémico (LES) y es uno de los criterios de clasificación del SLICC-2012. El objetivo del estudio fue verificar el desempeño del inmunoensayo quimioluminiscente (CLIA) QUANTA Flash dsDNA y compararlo con el método en uso de inmunofluorescencia indirecta en Crithidia luciliae (CLIFT). Se analizaron con ambos métodos 195 pacientes con diagnóstico de enfermedades del tejido conectivo y solicitud de anticuerpos anti-dsDNA. Se obtuvieron 38 sueros positivos, 133 negativos y 24 (12,3%) discordantes. Entre estos resultados discordantes, hubo 17 que correspondieron a pacientes con LES y se agruparon en 16 CLIA+/CLIFT- y 1 CLIA-/CLIFT+. Se verificó el desempeño para precisión siguiendo el protocolo EP15-A2 y la linealidad. Se estudió la concordancia mediante el coeficiente Kappa y la correlación con el coeficiente Rho de Spearman. Se observó mayor sensibilidad diagnóstica para CLIA. El grado de acuerdo fue moderado y se obtuvo buena correlación entre los valores cuantitativos de CLIA y los títulos de CLIFT. De acuerdo al buen desempeño encontrado y a los resultados discordantes analizados, la mejor estrategia para la implementación de CLIA sería utilizarla en combinación con CLIFT, lo que aumentaría la sensibilidad diagnóstica sin perder especificidad.

Abstract The detection of anti-dsDNA antibodies is useful for diagnosis and clinical monitoring of patients with systemic lupus erythematosus (SLE) and it is part of the classification criteria according to SLICC 2012. The purpose of this study was to verify the performance of the chemiluminescent immunoassay (CLIA) QUANTA Flash dsDNA and to compare it with the method currently in use, i.e the indirect immunofluorescence in Crithidia luciliae (CLIFT). One hundred and ninety five patients, who presented connective tissue diseases and required the study of anti-dsDNA antibodies, were analyzed. Thirty eight positive serum samples were obtained, 133 were negative and 24 (12.3%) in disagreement. Within the discordant results, there were 17 that corresponded to patients with SLE and they were grouped in 16 CLIA+/CLIFT- and 1 CLIA-/CLIFT+. The accuracy performance was assessed according to the EP15-A2 protocol and linearity. Concordance and correlation were calculated with the Kappa and Spearman's Rho coefficient, respectively. Based on the good performance observed and the discordant results analyzed, the best strategy for CLIA implementation would be to combine it with CLIFT, which would increase the diagnostic sensitivity without losing specificity.

Resumo A determinação dos anticorpos anti-dsDNA é de utilidade para o diagnóstico e seguimento clinico de pacientes com lúpus eritematoso sistêmico (LES) e é um dos critérios de classificação do SLICC 2012. O objetivo do estudo foi verificar o desempenho do imunoensaio quimioluminescente (CLIA) QUANTA Flash dsDNA e compará-lo com o método em uso imunofluorescência indireta em Crithidia luciliae (CLIFT). Foram analisados com os dois métodos, 195 pacientes com diagnóstico de doenças do tecido conjuntivo e solicitude de anticorpos anti-dsDNA. Os resultados foram agrupados em 38 soros positivos, 133 negativos e 24 (12,3%) discordantes. Entre esses resultados discordantes, 17 corresponderam a pacientes com LES e se agruparam em 16 CLIA+/CLIFT- e 1 CLIA-/CLIFT+. Foi verificado o desempenho para precisão seguindo o protocolo EP15-A2 e a linearidade. Foi estudada a concordância mediante o coeficiente Kappa e correlação com o coeficiente Rho de Spearman. Observou-se maior sensibilidade diagnóstica para CLIA. O grau de acordo foi moderado e boa correlação foi observada entre os valores quantitativos de CLIA e os títulos de CLIFT. Com base no bom desempenho encontrado e nos resultados discordantes analisados, a melhor estratégia para implementar o CLIA seria utilizá-lo em combinação com o CLIFT, o que aumentaria a sensibilidade do diagnóstico sem perder a especificidade.

Feline Leishmania spp. Infection in a Non-Endemic Area of Northern Italy.

Feline leishmaniosis (FeL) is an emerging vector-borne feline disease, with increasing numbers of cases reported and studies performed internationally. This study aimed to update the epidemiological status for FeL in stray cats in Milan, northern Italy; compare these results with previous studies in Northern Italy; and report clinicopathologic findings and coinfections in cats infected with Leishmania spp. A total of 117 cats were tested for L. infantum and retrovirus infection, hematological, and biochemical parameters. Demographic and clinical data were collected and FeL affected cats screened for selected coinfections. Overall, 10/117 (8.6%) cats tested positive for L. infantum: in five cats L. infantum DNA was found in popliteal lymph nodes and five were IFAT seropositive at titers from 1:80 to 1:160. Infected cats were concentrated in a specific area of Milan (p = 0.0154). No specific clinicopathologic abnormalities or retroviral infections were significantly linked to the infection, other than hypergammaglobulinemia (p = 0.0127). Seroreactivity to Anaplasma phagocytophilum, Chlamydophila felis, and Toxoplasma gondii was found in some infected cats. A high prevalence of FeL was found in a non-endemic area of northern Italy and future studies should continually monitor this data to understand whether these cases are imported or if Leishmania vectors are present in this area.

Serological, molecular and clinicopathological findings associated with Leishmania infantum infection in cats in Northern Italy.

OBJECTIVES: The aims of this study were to investigate the prevalence of Leishmania species infection in cats in Northern Italy and to evaluate the associations between infection and signalment and clinicopathological data. METHODS: The study was carried out in a veterinary university hospital from June to November 2017. Blood, urine, conjunctival swabs and hair were collected from all randomly selected cats. Leishmania species infection was evaluated using the indirect fluorescent antibody test (IFAT), setting a cut-off value of 1:80, and using real-time PCR on blood, conjunctival and hair samples. A complete blood count, serum chemistry profile, serum electrophoresis and urinalysis were also carried out. The cats were grouped on the basis of the results of the diagnostic criteria adopted in positive, negative and unconfirmed Leishmania cases. Non-parametric variables and continuous data were compared among the study groups using the χ2 test and the Mann-Whitney U-test, respectively. RESULTS: One hundred and fifty-two cats were included. Nineteen of the 152 (12.5%) cats were positive (18/152 [11.8%] showed an IFAT titre of ⩾1:80 and 1/152 [0.7%] was real-time PCR-positive from a hair sample); 106/152 (69.7%) cats were negative; and 27/152 (17.8%) cats were unconfirmed for Leishmania species. Total proteins, beta2-globulin and gamma-globulin were significantly increased in the positive Leishmania group compared with the negative group. CONCLUSIONS AND RELEVANCE: The results of the present study demonstrated the spread of Leishmania infantum infection in cats in Northern Italy. Hyperproteinaemia and hypergammaglobulinaemia appeared to be significant clinicopathological abnormalities in this population of cats with L infantum infection.

Evaluation of the automated indirect immunofluorescence test for anti-dsDNA antibodies.

BACKGROUND: Anti-dsDNA antibody is a specific antibody in systemic lupus erythematosus (SLE). Indirect immunofluorescence test (IIFT) is a highly specific method in detecting anti-dsDNA antibody. The application of automated system has gained better consistency than manual operation. This study detected anti-dsDNA antibodies using EUROPattern Computer-aided immunofluorescence microscopy (EPA), and evaluated the performance of the automated system. METHODS: The sera of 96 patients with suspected SLE and 102 control patients were examined using IIFT. The consistency between the EPA and manual reading was analyzed. RESULTS: Analysis of 198 samples showed that the overall consistency of the negative/positive results between the EPA and manual reading was 94.95%. Based on the manual reading results, the sensitivity and specificity of EPA were 95.70% and 94.29%, respectively. The analysis of 57 samples with non-specific fluorescence showed that the overall consistency of the negative/positive results was 96.49%. The analysis of the antibody titer of 89 positive samples showed that the consistency between the EPA and manual reading was 97.75%. CONCLUSION: EPA was consistent with the manual reading with regard to qualitative reading and antibody titer. With low-exposure function, EPA could read samples with non-specific fluorescence. EPA was superior to manual reading in automation and standardization.

Retrospective analysis of vector-borne infections in dogs after travelling to endemic areas (2007-2018).

Canine vector-borne infections gained in importance in Germany due to growing tourist traffic, the increased import of dogs from abroad and the changing of climatic conditions. The Mediterranean region and southeastern Europe are geographical areas where pathogens such as Leishmania (L.) infantum, Hepatozoon (H.) canis, Ehrlichia (E.) canis, Anaplasma (A.) platys and Dirofilaria (D.) spp. are endemic. Meanwhile, Babesia (B.) spp. and A. phagocytophilum are present in central and western Europe. The objective of this retrospective study was to evaluate whether dogs were exposed to a corresponding risk of infection when travelling to regions in the Mediterranean area and southeastern Europe, which are endemic for these pathogens. Medical records and laboratory test results of 303 dogs that travelled to 14 countries endemic for the mentioned canine vector-borne pathogens and that were presented to the Small Animal Clinic at Freie Universität Berlin between 2007 and 2018 were retrospectively reviewed. A total of 1174 test results from external laboratories were descriptively analysed including 525 test results of direct and 649 of indirect determination methods. Overall, 13% of the tested dogs (40/303) were positive for at least one pathogen. Concurrent infections with two pathogens were detected in 1% of the dogs (4/303). The positive results were: E. canis 8% (18/231 dogs; Polymerase chain reaction [PCR] 3/73, indirect immunofluorescence test [IFAT] 18/209 dogs), L. infantum 5% (14/260 dogs; PCR 5/80, IFAT or enzyme linked immunosorbent assay [ELISA] 11/251 dogs), Babesia spp. 5% (11/232 dogs; Babesia spp. PCR 3/127, B. canis/vogeli IFAT or ELISA 8/160, B. gibsoni IFAT 2/22), Dirofilaria spp. 1% (1/133 dogs; D. immitis antigen-ELISA 1/117, microfilariae PCR 0/16, Knott´s test 0/69 dogs). None of the dogs has been tested positive in a combined Babesia spp./Hepatozoon spp. PCR test (0/15 dogs) and for H. canis (0/17 dogs; PCR) or A. platys (0/11 dogs; PCR). There is a substantial risk for dogs travelling to areas endemic for vector-borne pathogens even with limited time of exposure to get infected. The data indicates the importance of owner education and prophylactic measurements against vector-borne infections in dogs travelling to endemic areas.

Reliable Serological Testing for the Diagnosis of Emerging Infectious Diseases.

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.There are two pillars to diagnosis: direct pathogen detection and the determination of specific antibodies. Serological tests provide a longer diagnostic window than direct methods, and are suitable for diagnosing acute and past infections, for disease surveillance and for vaccination monitoring. ELISA and indirect immunofluorescence test (IIFT) systems based on optimized antigens enable sensitive and specific detection of antibodies against ZIKV, DENV and CHIKV in patient serum or plasma. In recent years, Euroimmun (Lübeck, Germany) has developed numerous test systems for the serological diagnosis of (re-)emerging diseases, including a very sensitive and specific anti-ZIKV ELISA.

Antibodies for Rickettsia spp. in patients with negative serology for dengue virus, leptospirosis, and meningococcal disease in municipalities of São Paulo State, Brazil

Abstract INTRODUCTION Brazilian spotted fever is an infectious disease with a high mortality rate if not treated early. Differential diagnosis is difficult, as the first clinical signs are non-specific and can be confused with other diseases. The aim of the study was to investigate evidence of infection with Rickettsia rickettsii and Rickettsia parkeri in negative sera samples, collected in 2014, from patients with suspected leptospirosis, dengue fever, and meningococcal disease in Atibaia and Bragança Paulista municipalities of the State of São Paulo. METHODS The samples stored at the Institute Adolfo Lutz in Campinas were tested using an indirect immunofluorescence assay (IFA) with IgG and IgM against R. rickettsii and R. parkeri. Real-time polymerase chain reaction (PCR) testing was performed for the sera samples of patients who died (n = 3), those with initial suspicion of meningococcal disease (n = 6), and those with positive IFA results. RESULTS Of 258 samples from Bragança Paulista, 4 (1.6%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. Of 155 samples from Atibaia, 2 (1.3%) were positive, with IgG titers of 1:64 and 1:128 against R. rickettsii and R. parkeri, respectively. No sample showed positive PCR results. CONCLUSIONS This serological investigation suggests there is evidence of exposure to Rickettsia spp. in residents of areas that have environmental conditions favorable to the spread of bacteria, in which Brazilian spotted fever incidence was not previously confirmed.

Screening red foxes (Vulpes vulpes) for possible viral causes of encephalitis.

BACKGROUND: Next to various known infectious and non-infectious causes, the aetiology of non-suppurative encephalitis in red foxes (Vulpes vulpes) often remains unclear. Known causes in foxes imply rabies, canine distemper, toxoplasmosis, Aujeszky's disease, as well as parvovirus, adenovirus, circovirus and flavivirus infections. In this study, particular attention was paid on bornaviruses, since red foxes are predators of bicoloured white-toothed shrews, a reservoir of Borna disease virus 1 (BoDV-1). In addition, foxes are known to be highly susceptible for viruses of the order Mononegavirales. METHODS: Analyses for the presence of anti-BoDV-1 antibodies, BoDV-1-RNA and antigen were performed on 225 blood and 59 brain samples, from a total of 232 red foxes. Foxes originated from BoDV-1 endemic and non-endemic German areas. Additional investigations for the presence of rabies, canine distemper, toxoplasmosis, Aujeszky's disease, parvovirus, adenovirus and flavivirus infections were carried out on 16 red foxes with non-suppurative (meningo-) encephalitis. A metagenomic analysis was used on three representative brain samples displaying encephalitis. RESULTS: Among 225 foxes, 37 displayed anti-BoDV-1 antibodies with titres ranging between 1:40 and 1:2560, regardless of geographic origin. In 6 out of 16 foxes with encephalitis, canine distemper virus was detected. No evidence of any of the other investigated agents was found in the 16 fox brains with encephalitis. Metagenomics revealed no infectious agents, except for one already known canine distemper case. CONCLUSION: Red foxes can exhibit BoDV-1 specific antibodies without association with geographic origin or encephalitis due to bornavirus infection. The encephalitis pattern was highly conspicuous for a viral infection, but remained unclear in 10 out of 16 foxes. Thus, presently unknown infectious and non-infectious causes need to be considered and further investigated, especially since foxes also tend to occur in human proximity.

Long-lasting Localized Pemphigus Vulgaris without Detectable Serum Autoantibodies Against Desmoglein 3 and Desmoglein 1.

Pemphigus vulgaris (PV) is an autoimmune blistering disease elicited by anti-desmoglein (DsG) 3 antibody. Although skin lesions tend to be distributed over the entire body, in some patients, they are confined to a restricted area. We report two patients who presented with long-lasting localized PV without detectable anti-DsG antibodies after suffering antibody-positive systemic PV. Initial treatment with prednisolone (PSL) was successful in both patients, but a local relapse occurred on the cheek or lower lip after a reduction in the PSL dose. Biopsy of the localized lesions showed suprabasal acantholysis; no serum DsG antibodies were found. Local immunosuppression therapy was effective in both patients. Based on our findings, we suggest that localized PV without detectable antibodies can develop after systemic PV.

Serological Evidence of Scrub Typhus among Cases of PUO in the Kashmir Valley- A Hospital Based Study.

INTRODUCTION: Rickettsial infections are being increasingly recognized as a cause of acute febrile illnesses and should be considered a distinct possibility in patients presenting with suggestive clinical features. Their diagnosis remains a challenge in a country like ours where tests like immunofluorescence assay cannot be routinely done. Results of serological tests, when correlated with patients clinical profile can aid in the timely diagnosis of scrub typhus. AIM: To find out the extent to which scrub typhus contributes to Pyrexia of Unknown Origin (PUO) in patients admitted to or attending the OPD of our hospital using simple tests like Weil-Felix Agglutination Test (WFT) and Enzyme Linked Immunosorbent Assay (ELISA). MATERIALS AND METHODS: A cross-sectional study was carried out in the Department of Microbiology, Government Medical College and Hospital, Srinagar, over a period of eight months (1(st) March to 31(st) October 2015). Serum samples from patients suffering from Pyrexia of Unknown Origin (PUO) were processed for the detection of Scrub typhus. A total of 162 samples were included in the study. These were subjected to WFT using OX-K strain. The serum samples were diluted 1/20 to 1/640 and a titre of ≥ 1:160 was considered as positive. The samples were also tested for IgM and IgG antibodies for scrub typhus by ELISA and tube agglutination test was done to detect typhoid fever and brucellosis. RESULTS: Of the 162 serum samples tested 22.8% tested positive scrub typhus by WFT. IgM ELISA and IgG was positive in 8 (4.9%) and 15 (9.3%) samples respectively. Sensitivity, specificity, positive and negative predictive values of WFT; taking IgM ELISA as a reference standard were 75%, 79.9%, 16.2% and 98.4% respectively. CONCLUSION: Scrub typhus is prevalent in our state and the results of WFT supplemented by those of ELISA can aid in its diagnosis. However the results of these tests should always be regarded in light of the clinical condition of the patient.

Validation of a phospholipase A2 receptor antibody ELISA in an Australian cohort with membranous glomerulonephritis.

A commercial PLA2R Ab ELISA was validated by examining its ability to distinguish primary from secondary membranous nephropathy, correlating results with clinical markers of disease activity, and comparing its performance with an indirect immunofluorescence test (IIFT). PLA2R Ab levels were measured in 77 patients with biopsy proven membranous nephropathy, divided into either idiopathic (n = 61) or secondary groups (n = 6). In the idiopathic group, measures of contemporaneous disease activity (proteinuria, serum creatinine) were compared between seropositive and seronegative subjects. ELISA values were then compared with semi-quantitative results from an IIFT using PLA2R transfected HEK293 cells as substrate. The PLA2R Ab ELISA was positive in only 15 of 61 (25%) patients with idiopathic membranous nephropathy (IMN), but there was a significant negative relationship with time since diagnosis. Thus, in a subgroup of patients diagnosed within 6 months of analysis, the sensitivity was 6/15 (55%), rising to 6/8 (75%) in those recently-diagnosed patients who had not been treated. In the entire cohort, there was a significant positive correlation between ELISA values and degree of proteinuria, but our analysis did not control for variation of both variables with time. The PLA2R Ab ELISA also showed very high agreement with IIFT (96%). Therefore, the PLA2R Ab ELISA is a highly specific test for distinguishing primary from secondary membranous nephropathy that is most sensitive in newly diagnosed patients who have not received immunosuppression. Antibody levels correlated with degree of proteinuria, but this relationship was not shown to be independent of time. Both IIFT and ELISA platforms performed comparably.

Usefulness of a rapid immuno-migration test for the detection of canine monocytic ehrlichiosis in Africa.

A rapid immuno-migration test for the serological detection of canine monocytic ehrlichiosis, Witness® Ehrlichia (WE) (Zoetis, France), was evaluated in 528 serum samples from dogs living in endemic areas of West and East Africa: Senegal (N=208), Ivory Coast (N=7), Sudan (N=27), and Djibouti (N=286). Of these dogs, 200 were French military working dogs (MWD) temporarily residing in Africa. The WE test results were compared with those obtained by indirect immunofluorescence (IFA). The sensitivity of WE was 97% [94.2, 98.7] with a specificity of 100% [98.6, 100]. In MWD, the seroprevalence (IFA) was 7%; in native dogs, it reached 77.1%. This significant difference can be explained by prophylactic measures from which MWD benefit. The WE test represents a simple, fast and reliable test for the detection of anti-Ehrlichia canis antibodies. Its implementation for the diagnosis of clinical cases has been validated in the field, and its use allows easy detection of asymptomatic dogs that may be carriers of E. canis.

Anti-neuronal autoantibodies: Current diagnostic challenges.

The spectrum of neurological autoimmune diseases has expanded substantially in the last 15 years due to the discovery of new anti-neuronal antibodies. There are at present numerous technical challenges for developing and improving standardized serological test systems for the detection of these autoantibodies, some of which occur very rarely. In particular, the determination of autoantibodies against complex cell surface structures generally requires authentically presented target antigens. Finally, research into syndrome associations benefits from multiplex analyses and accelerates the understanding of the complex autoimmune processes, forming an important basis for the development of novel therapy concepts.

Autoantibodies against exocrine pancreas in Crohn's disease are directed against two antigens: the glycoproteins CUZD1 and GP2.

BACKGROUND: Autoantibodies against exocrine pancreas (PAb) have been reported to be pathognomonic markers of Crohn's disease (CD). Recently, the glycoprotein GP2 has been proposed as the exclusive target for PAb but two equally prevalent binding patterns can be observed in the indirect immunofluorescence test (IIFT) using cryosections of human pancreas: a reticulogranular and a droplet pattern. AIM: To identify autoantigens corresponding to the staining patterns. METHODS: Different lectins were screened for their ability to immobilize PAb-reactive glycoproteins from cell free human pancreas. The glycoproteins were then purified via UEA-I affinity chromatography and identified by mass spectrometry. The two candidate autoantigens were separately expressed in HEK293 cells, and the recombinant cells applied as substrates in IIFT to analyze sera from 96 patients with CD, 89 controls and hybridoma supernatants during the generation of murine monoclonal antibodies. RESULTS: The UEA-I eluate was able to neutralize PAb reactivity of both patterns in IIFT. It contained two major constituents which were identified as the glycoproteins CUZD1 and GP2. With the recombinant cells, 35.4% of the CD patients exhibited positive reactions (CUZD1 alone 19.8%, GP2 alone 9.4%, and both antigens 6.2%). The reaction with the CUZD1 expressing cells was strictly correlated to the reticulogranular pattern, whereas the antibodies causing the droplet pattern stained the GP2 expressing cells. Antigen-capture ELISA using the newly generated monoclonal antibodies against CUZD1 and GP2 verified this relationship. CONCLUSIONS: The concordant reactivities of the different platforms can be regarded as a proof for the authenticity of the two identified autoantigens.

Two Cases of Autoimmune Neutropenia of Infancy / 임상소아혈액종양

Autoimmune neutropenia of infancy (AIN) is caused by increased peripheral destruction of neutrophils as a result of antibodies in patients' blood that are directed against their own neutrophils. Due to non-specific symptoms, benign clinical courses, and cumbersome diagnostic tests, AIN are commonly undetected. Antineutrophil antibody test for diagnosis of AIN has recently become available. Compared to its relatively lower absolute neutrophil count (ANC), the clinical course of AIN is mostly benign. Therefore, although treatment is not usually necessary for AIN, it is applicable in order to rule out other significant diseases, such as severe congenital neutropenia (SCN), which can be transformed to myelodysplastic syndrome or acute myelocytic leukemia. For this reason, several treatments can be used for neutropenia: granulocyte-colony stimulating factor (G-CSF) for SCN, trimethoprim and sulfamethoxazole (TMP-SMX) for prophylaxis. Here we report on two cases of AIN confirmed by indirect immunofluorescence test using flow cytometry.

Two Cases of Autoimmune Neutropenia of Infancy / 임상소아혈액종양

Autoimmune neutropenia of infancy (AIN) is caused by increased peripheral destruction of neutrophils as a result of antibodies in patients' blood that are directed against their own neutrophils. Due to non-specific symptoms, benign clinical courses, and cumbersome diagnostic tests, AIN are commonly undetected. Antineutrophil antibody test for diagnosis of AIN has recently become available. Compared to its relatively lower absolute neutrophil count (ANC), the clinical course of AIN is mostly benign. Therefore, although treatment is not usually necessary for AIN, it is applicable in order to rule out other significant diseases, such as severe congenital neutropenia (SCN), which can be transformed to myelodysplastic syndrome or acute myelocytic leukemia. For this reason, several treatments can be used for neutropenia: granulocyte-colony stimulating factor (G-CSF) for SCN, trimethoprim and sulfamethoxazole (TMP-SMX) for prophylaxis. Here we report on two cases of AIN confirmed by indirect immunofluorescence test using flow cytometry.

Autoimmune neutropenia due to antineutrophil antibodies in a patient with primary sclerosing cholangitis.

Autoimmune neutropenia (AIN) is defined as a decrease in the circulating absolute neutrophil count (ANC) to less than 1500/µl caused by serum antineutrophil antibodies. Secondary AIN is associated with various autoimmune diseases. Herein we present the case of a patient with primary sclerosing cholangitis (PSC) who developed secondary AIN. A 19-year-old man was admitted due to liver injury, and a diagnosis of PSC was established by cholangiogram and liver biopsy. Severe neutropenia, with the ANC down to 130/µl, developed during his hospital course. No medications had been given at that time and bone marrow aspiration revealed no abnormality. Therefore we suspected secondary AIN as a causative etiology and examined whether antineutrophil antibodies were detectable in the patient's sera by flow cytometric analysis of the granulocyte indirect immunofluorescence test. We found that antineutrophil antibody was strongly positive on admission, and the titer decreased along with recovery from neutropenia. This is the first reported case of a patient with PSC who developed AIN, with detection of serum antineutrophil antibodies.